Thirty temperature-sensitive mutants of encephalomyocarditis (EMC) virus have been isolated and partially characterized. Eleven of these mutants have a defect in the proteolytic processing of the viral protein precursors when grown at the nonpermissive temperature of 39.5 C. One of the eleven appears to have a defect only in the proteolytic cleavage step which occurs during virion maturation and which normally produces capsid proteins beta and delta from protein epsilon. Further study of this mutant is proposed to learn more about the maturation of EMC virus. The remaining eleven mutants are similar in that cleavage of the capsid precursor proteins A and B occurs very poorly. It is unknown if these cleavage defects are due to defects in the precursor proteins altering the cleavage sites or to defects in a viral-specific cleavage enzyme or viral-specific protein factor involved in cleavage. Several approaches are proposed to help solve this problem. 1) In vitro cleavage reactions will be performed at 39.5 C and at the permissive temperature of 33 C with various cytoplasmic extracts from EMC mutant and wild type infected cells. 2) Viral-specific proteins from HeLa cells infected with cleavage-defective mutants will be subjected to two-dimensional electrophoresis to determine if any specific protein has a charge due to a mutation and is thus possibly involved in cleavage. 3) Complementation of cleavage-defective mutants with EMC defective interfering (DI) particles will be performed at 39.5 C to determine if the necessary functional cleavage enzymes are present in the coinfected cells. 4) Mutants of EMC virus will be sought which are capable of growth in the presence of a protease inhibitor which would normally block at least some viral-specific cleavages. 5) EMC viral-specific proteins made in different cell lines will be compared by one- and two-dimensional electrophoresis to see if the proteins are similar regardless of the source of the host cell proteolytic enzymes.